The purpose of this project is to elucidate the mechanisms and principles of prevention and treatment of chronic pain syndromes. Our main current focus is the examination of pain candidate genes in cohorts of pain patients and controls. This year we collected DNA from 314 patients with pain from herniated lumbar disc compressing the nerve root who had been closely followed for 10 years in the Maine Lumbar Spine Study. We chose 20 candidate genes from the pain neuroscience literature by a process that can be applied to any area for initial association studies: We compiled a list of about 200 putative pain-mediating molecules by searching recent pain research abstracts, review articles, and textbooks. We then searched PubMed for each molecule, using the terms ?[molecule] and human and polymorphism? and rated each polymorphism of the molecule on a 0-3 point scale for (1) strength of evidence for involvement in pain processing, particularly for persistent pain after nerve injury; (2) population frequency; and (3) strength of clinical or in vitro evidence that the polymorphism changes function. The highest scoring 20 candidate genes included polymorphisms of genes for cytokine genes and their receptors, pain-related excitatory and inhibitory neurotransmitters and receptors, and growth factors. We then prepared dense haplotype assays for these genes. Our preliminary findings are that patients who carry an interleukin-1 beta polymorphism associated with hypersecretion of that inflammatory cytokine, which had been our highest priority candidate gene, are significantly more likely to have persistent pain in the two years after spine surgery. We have also found that a polymorphism that decreases secretion of the anti-inflammatory cytokine IL-13 is also associated with greater risk of persistent post-surgical sciatica. We are exploring this finding with additional genotyping and haplotype analysis and in vitro cell function studies of the polymorphisms associated with greater pain. We are also exploring genetic risk factors for temporomandibular pain in a longitudinal study of 220 normal women in collaboration with Luda Diatchenko and William Maixner at the University of North Carolina Dental School. We identified three genetic variants (haplotypes) of COMT that we designated as low pain sensitive (LPS), average pain sensitive (APS), and high pain sensitive (HPS) that account for 11% of variability in pain perception. The presence of even a single LPS haplotype diminishes, by as much as 2.3 times, the risk of developing myogenous temporomandibular joint disorder (TMD), a common musculoskeletal pain condition. The LPS haplotype produces much higher levels of COMT enzymatic activity compared to the APS or HPS haplotypes. Thus, three major haplotypes determine the levels of COMT activity that inversely correlate with pain sensitivity and the risk of developing TMD, and possibly other related chronic pain conditions. This is the first study that has identified a genetic polymorphism associated with the risk of developing a chronic pain condition. In a collaborative study with NIMH and Howard University, we showed that a common deletion in the alpha-2C adrenergic receptor is associated with excessive release of the catecholamines norarenaline and adrenaline, which may account for some of the higher risk of hypertension, congestive heart failure, and depression in African-Americans. Twenty-nine healthy African-Americans genotyped for alpha-2 adrenergic receptor subtype polymorphisms underwent tritiated-noradrenaline and adrenaline intravenous infusion and arterial blood sampling for measurements of rates of entry of endogenous noradrenaline and adrenaline into arterial plasma (total body spillovers) by the tracer dilution technique. Eleven subjects were homozygotes for the alpha 2C deletion polymorphism, 9 intravenous infusion of the alpha-2-adrenergic receptorr antagonnist, yohimbine. Administration of yohimbine produced larger, more sustained increments in noradrenaline spillover, heart rate, and anxiety in homozygotes for the receptor molecule deletion than in the other groups. In another study in Caucasians, we and our NIMH collaborators observed an association of this same common deletion in the alpha 2C adrenergic receptor gene with the occurrence of recurrent depression. In other studies, we delineated the haplotype block structure of our 25 pain candidate genes in Caucasian, African-American, and American Indian populations. We are also continuing clinical trials of single drugs, drug combinations, and alternative medicine techniques in chronic lumbar radiculopathy pain; and with NCCAM?s Suzan Khoromi and Marc Blackman have initiated studies of the effects of chronic pain, opioid therapy, and placebo treatment upon endocrine function. An interesting interim result is that in the first 10 patients completing a four-period crossover study, a combination of nortriptyline and morphine, titrated to dose-limiting side effects, provides more pain relief than either single treatment alone. If confirmed by the outcomes of the 35 other patients still in treatment, this will be one of the first studies suggesting one can exceed the current ?barrier? of a mean of 30% pain relief seen with single agents in pain from nerve injury.